Western blotting is a ubiquitous analytical technique for identifying and quantifying specific proteins in a complex mixture. In the technique, gel electrophoresis is used to separate proteins in a gel based on properties such as tertiary structure, molecular weight, isoelectric point, polypeptide length, or electrical charge. Once separated, the proteins are then transferred from the gel to a membrane—typically made of nitrocellulose, nylon, or polyvinylidene fluoride (PVDF)—that binds proteins non-specifically. A commonly used method for carrying out this transfer is electroblotting, in which an electrical current is used to pull proteins from the gel into the membrane. The membrane is then stained with probes specific for the proteins being targeted, allowing the location and amounts of these proteins to be detected.
Capillary electrophoresis provides an alternative to the gel electrophoresis separation associated with western blotting and other biotechnology procedures. In capillary electrophoresis, materials such as proteins are separated electrokinetically, as in gel electrophoresis, but with much smaller required volumes. The capillaries used in this technique are typified by diameters smaller than one millimeter and are in some instances incorporated into microfluidic or nanofluidic devices.
Previous work has demonstrated the benefits of applying microfluidic devices to Western blotting of proteins (Jin et al. 2013 Anal. Chem. 85:6073). These devices electrically transfer separated proteins to a blotting surface that is itself the terminating electrode. (See, e.g., U.S. Pat. No. 9,182,371). This electrical field blotting approach requires continuous electrical contact from a separation device to the surface. As a result, the surface must be electrically conductive (e.g., a wet membrane on metal platen).
In electric field blotting, proteins migrate toward the surface via electrophoresis. Since the cross-sectional area of the current flow abruptly increases upon exiting the separation device, the electric field abruptly diminishes. Also, since the surface is typically wet, a large meniscus tends to form around the point of contact between the separation device and the surface. This large meniscus can comprise recirculation zones in which analytes such as proteins can be trapped and mixed, reducing the resolution of separation. Furthermore, the electrical field blotting force is only applied while the separation device is above the analyte. If a surface and separation device move to a different position relative to one another, the electrical force is removed and only diffusion forces cause the analyte to become immobilized in the surface membrane.